In certain clinical diagnostic analyzers, dry analytical slide elements are utilized for immunorate chemistry assays. Examples of such elements include those manufactured by Eastman Kodak Company under the registered trademark EKTACHEM.RTM..
Typical dry chemistry test elements, have a porous spreading layer containing antibodies immobilized therein that act as adsorption sites for an analyte of interest, such as a drug (D) and a chemical label (L); for example, a conjugate of the drug and a detectable species such as an enzyme. Typically, a sample containing drug (D) is applied to the spreading layer as a spot from a point source, such as a pipette tip. The sample quickly penetrates the pores of the spreading layer and simultaneously reconstitutes (dissolves) the chemical label (L) which has been previously coated onto the top of the spreading layer or applied as a separate solution. Alternatively, the chemical label can be added to the sample and the mixture applied to the element. Ideally, the label L becomes uniformly distributed in the liquid sample regardless of how it is applied. Thus, when the sample application process is complete, the spreading layer is saturated with a uniform layer of D & L. Because immobilized antibody is present throughout the spreading layer, D & L begin to bind to these adsorption sites. In order to detect the level of D present in the sample, there must be a competition between D & L for the limited number of antibody adsorption sites in the layer. That is, the amount of D is determined from its relative ability to compete against L for adsorption sites available in the spreading layer.
In an alternative type of immunological assay, commonly referred to as a sandwich assay, an antibody is contacted with a sample containing an analyte or drug (D) to cause the analyte to bind to the antibody. This complex is then contacted with a labeled antibody which reacts with the bound analyte. The amount of bound labeled antibody is thus directly proportional to the amount of bound analyte.
To measure D's success in competing for antibody sites, the amount of L bound to antibody is measured. Unfortunately, the differences between bound and unbound label (L) can not be distinguished, other than the fact that bound label is immobilized. Therefore, to measure the amount of L bound to the immobilized antibody, the L in solution has to be washed away from the region to be read by the detection device, such as an optical reflectometer, to determine the amount of D present by sensing the presence of L. This defines the need for a wash step or operation included in the assay procedure to facilitate the removal of unbound label from the detection read area of a slide element. Typically, this wash step is carried out in conventional immunoassays by applying about 30 to 100 microliters of wash liquid to the center of the area where the sample was spotted. As the wash liquid flows into the spreading layer, it displaces (pushes) the sample fluid containing dissolved and unbound L outward.
It has been found that conventional dispensing techniques, in which wash liquid is metered or dispensed directly to the center of the sample spot, fail to follow the idealized format above. Instead, wash liquid enters and traverses along the test element top surface and unbound label is washed out, primarily in the annular region of the spreading layer just below the contact line of the fluid meniscus (lens) created by the wash fluid on the top surface of the spreading layer. Consequently, this has been found to leave a significant fraction of unbound L in the center of the spot.
An improved wash technique is described in commonly assigned U.S. Ser. No. 880,902, filed 6 May 1992, now abandoned, in which wash liquid is applied by conventional means at a position in between the center of the sample spot and the inner periphery of the area wetted by the sample. Applying wash liquid in this manner allows the central portion of the original sample spot to be adequately washed of unbound label, while excess label at the periphery will not be redistributed back towards the center. In this way, a smaller volume of wash fluid (typically, about 10 to 13 uL) is required to push unbound L from the detection read area of the test element.
In any event, it is known that an insufficient wash volume, regardless of where it is applied, will fail to push the unbound label from the detection read area of an element, thereby producing undesirable and inaccurate test results.
Current means for verifying a wash operation include the use of wetness detectors which can generally sense the placement of a liquid onto the dried chemistry portion of a test slide element. This type of apparatus, an example of which is described in U.S. Pat. No. 5,084,620, is quite useful for indicating whether a premature wetness of a dried chemistry test element has occurred prior to sample dispensing, but inadequate otherwise in that it merely provides general qualitative determinations on a prewetted element, and not quantitative determinations.
Alternately, pressure transducers can be used to describe the presence of liquid on a test element. However, these devices are similarly limited and can only provide general information that a liquid has been applied and are not particularly effective to verify the success or failure of a wash sequence.
Therefore, there is a need to provide a means for verifying a wash process which will provide increased confidence that an adequate wash volume has been dispensed to displace unbound label from the read area of the slide element.